Promotion of somatic embryogenesis in plants by Wuschel gene expression

ABSTRACT

The present invention relates to methods for promoting somatic embryogenesis from a tissue or organ of a plant, by overexpressing a  Wuschel  gene in said tissue or organ. In one embodiment, such overexpression can be used as a silent selectable marker for transgenic plants. In another embodiment, such expression can be used to confer apomixis to a plant. In another embodiment, such overexpression can be used to create haploid plants, which can be used to produce dihaploid plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a division of U.S. patent application Ser.No. 12/722,981 filed 12 Mar. 2010, which in turn is a division of U.S.patent application Ser. No. 10/956,120 filed 4 Oct. 2004, now U.S. Pat.No. 7,700,829, which in turn is a division of U.S. patent applicationSer. No. 09/984,274 filed 29 Oct. 2001, now abandoned. Each applicationis incorporated herein by reference.

BACKGROUND OF THE INVENTION

Somatic embryogenesis is a unique pathway for asexual propagation orsomatic cloning in plants. The developmental process of somaticembryogenesis shares considerable similarity with that of zygoticembryogenesis (Zimmerman, 1993; Mordhorst et al., 1997) and this islikely due to the conservation in the underpinning cellular andmolecular mechanisms between the two processes. Therefore, somaticembryogenesis provides an attractive model system for studying zygoticembryogenesis, particularly because zygotic embryos are encased bymaternal tissues and difficult to access by biochemical and moleculartools. Moreover, in biotechnological applications, most economicallyimportant crop as well as non-crop plants are regenerated via somaticembryogenesis.

In contrast to organogenesis, which requires a high cytokinin to auxinratio (Skoog and Miller, 1957; Sugiyama, 1999; Sugiyama, 2000), somaticembryogenesis does not require any external cytokinins, but rather isdependent on high concentrations of 2,4-D (Zimmerman, 1993; Mordhorst etal., 1997; Sugiyama, 2000), a synthetic chemical that has long been usedas a functional analog of auxin. It is generally believed that somaticembryogenesis is mediated by a signaling cascade triggered by externalauxin or 2,4-D (Zimmerman, 1993; Mordhorst et al., 1997; Schmidt et al.,1997). However, very little is known about the signal transductionpathway, particularly the molecular mechanism involved in the transitionof a vegetative cell to an embryogenic competent cell.

During the last two decades, considerable efforts have been made toidentify genes with altered expression patterns during somaticembryogenesis (Schmidt et al., 1997; Lin et al., 1996; Thomas, 1993).Most of these genes, however, are up-regulated only in latedevelopmental stages, suggesting that they do not play a direct role inthe vegetative-to-embryogenic transition. Thus far, the only exceptionis the carrot Somatic Embryogenesis Receptor-like Kinase (SERK) gene theexpression of which appears to mark the vegetative-to-embryogenictransition; however, its function remains unclear (Schmidt et al.,1997). An additional molecular approach was attempted by manipulatingcertain embryo-specific genes. The Arabidopsis Leafy cotyledon 1 (LEC1)gene, encoding a subunit of the HAP heterotrimeric transcription factorcomplex (HAP3), has been proposed as a key regulator for embryonicidentity (Lotan et al., 1998). Mutations in the LEC1 locus result indefective embryo maturation as well as the conversion of cotyledons intotrue-leaf-like structures (Lotan et al., 1998; Meinke, 1992; Meinke etal., 1994). Constitutive overexpression of LEC1 leads to severelyabnormal plant growth and development with occasional formation ofsomatic embryo-like structures (Lotan et al., 1998). The developmentalfate of these embryo-like structures, however, remained unknown due tothe lethality of LEC1 overexpression.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated herein byreference, and for convenience, are referenced by author and date in thetext and respectively grouped in the appended List of References.

SUMMARY OF THE INVENTION

One aspect of the present invention is a method to promote somaticembryogenesis from a tissue or organ of a plant, said method comprisingoverexpressing a Wuschel gene in said tissue or organ.

A second aspect of the invention is a method to generate somatic plantembryos wherein said method comprises overexpressing a Wuschel gene in atissue or organ of a plant.

Another aspect of the invention is a method for generating shoots from atissue or organ of a plant, said method comprising overexpressing aWuschel gene in said tissue or organ.

Yet another aspect of the invention is a method of selecting plantstransformed with a vector comprising a silent selectable marker whereinthe marker is a Wuschel gene.

Another object of the invention is a method of producing an apomicticplant line.

Another object of the invention is a method of producing haploid plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the XVE activation tagging vectorpER16.

FIGS. 2A-F illustrate the pga6 gain-of-function mutant phenotype. Rootexplants derived from pga6 seedlings were cultured on the non-inductiveSCM (SCM minus 17-(β-estradiol) for 20 days (FIG. 2A); or on theinductive SCM for 10 days (FIG. 2B), 20 days (FIG. 2C), or 30 days (FIG.2D). FIG. 2E shows an enlarged view of a germinating somatic embryoisolated from the explant shown in (FIG. 2D). FIG. 2F shows agerminating seedling derived from a somatic embryo grown on MS medium(45 days). Scale bar, 100 μm for FIGS. 2A and 2E; 1 mm, for FIGS. 2B,2C, 2D and 2F.

FIGS. 3A-D are electron microscopic analyses showing somaticembryogenesis in pga6 mutant explants (culturing conditions wereidentical to those shown in FIGS. 2A-F). FIG. 3A shows a pre-embryostage before the first embryonic cell division (arrows) and a two-cellstage after the first asymmetric division with a smaller apical cell (A)and a larger basal (B) cell. FIG. 3B shows embryos at the globular (G)and the early heart (H) stages. FIG. 3C shows a germinating embryo. C:cotyledon; H: hypocotyl. FIG. 3D shows an abnormal somatic embryo withthree cotyledons (C) anchored on the hypocotyl (H). Scale bar, 10 μm forFIG. 3A; 100 μm for FIGS. 3B-D.

FIGS. 4A-H illustrate phytohormone-independent somatic embryo formationcaused by the pga6 gain-of-function mutation. FIG. 4A is an overview ofpga6 mutant seedlings germinated and grown on MS medium (first seedlingfrom the left) or the inductive MS medium (MST: 5 μM 17-β-estradiol) for7 days. FIGS. 4B-D show pga6 seedlings that were cultured on theinductive MS medium for 10 days (FIG. 4B), 14 days (FIG. 4C) or 30 days(FIG. 4D). FIGS. 4E-F show seven-day-old pga6 seedlings germinated andgrown on MS medium which were transferred onto an inductive MS mediumand cultured for 5 (FIG. 4E) or 10 (FIG. 4F) days. FIG. 4G shows pga6root explants which were cultured on the inductive MS medium for 20days. FIG. 4H is an enlarged view of FIG. 4G. Scale bar, 1 mm.

FIGS. 5A-D show that the pga6 phenotype is due to the inducer-dependentoverexpression of WUS. FIG. 5A is a schematic diagram illustrating theinsertion site of the T-DNA upstream of the WUS gene (not shown toscale). Arrows indicate the directions of transcription. FIG. 5B showspga6 seeds (T2, homozygous) which were germinated and grown on MS mediumsupplemented with various concentrations of the inducer as indicated.Ten-day old seedlings are shown. The scale bar represents 1 mm. FIG. 5Cshows the expression of PGA6/WUS induced by different concentrations ofthe inducer. Ten-day-old pga6 seedlings were germinated and grown on MSmedium and transferred to an MS medium containing various concentrationsof 17-β-estradiol as indicated and were cultured for an additional 16hours before total RNA extraction. Five μg total RNA were used forNorthern blot analysis using a WUS cDNA fragment as a probe. Positionsof two RNA molecular weight markers are indicated at the right (GIBCOBRL, catalog number, 15620-016). FIG. 5D shows ethidium bromide stainingof the gel as a control for RNA loading and transfer.

FIGS. 6A-H are photographs showing that 35S- or XVE-controlledoverexpression of WUS cDNA phenocopies the pga6 phenotype. FIG. 6A showsembryogenic callus and FIG. 6B shows somatic embryo formation from roottips of XVE-WUS cDNA T2 seedlings grown for 15 days in A mediumsupplemented with 17-β-estradiol (10 μM). FIGS. 6C-H show 15 day-old T135S::WUS seedling phenotypes. FIG. 6C shows the tips of the roots areenlarged and show an embryo-like structure. FIG. 6D shows theadventitious root tip. FIG. 6E shows that WUS overexpression inducesboth organogenesis and embryogenesis from the root. FIG. 6F shows detailof early embryo structure formation. FIG. 6G shows the shoot apicalmeristem is dramatically altered and, besides forming lateral organswith altered shaped, givers rise to adventitious shoots and somaticembryos. FIG. 6H shows the entire shoot apical meristem expands andlateral organs transform into meristematic tissues. Scale bar is 1 mm.

FIGS. 7A-C are Northern blots of RNA from root explants prepared frompga6 seedlings cultured on the screening medium (SCM) for differenttimes as indicated. On day 28, when somatic embryos were apparent,cultures were transferred onto a freshly-prepared SCM or control medium(SCM without the inducer) and incubated for an additional day (28+1) ortwo days (28+2). Five micrograms of total RNA, prepared from the frozenmaterials, were analyzed by Northern blotting using WUS (FIG. 7A) andLEC1 (FIG. 7B) cDNA as probes. The blot was rehybridized with an actincDNA probe (FIG. 7C) to ensure that equal amounts of RNA were loaded.

FIGS. 8A-B illustrate formation of somatic embryos from isolated zygoticembryos of PGA6 transgenic plants grown in the presence (FIG. 8A) orabsence (FIG. 8B) of an inducer of PGA6.

DETAILED DESCRIPTION OF THE INVENTION

To dissect the signaling pathway during somatic embryogenesis, we haveemployed a genetic approach to identify gain-of-function mutations thatcan promote embryogenic callus formation from Arabidopsis root explants.Arabidopsis thaliana is known to be a species difficult for somaticembryogenesis. Thus far, embryogenic calli could only be induced fromimmature embryos of wild-type (WT) plants or from the primordia timing(pt) mutant plant (Wu et al., 1992; Mordhorst et al., 1998; andreferences therein). Therefore, Arabidopsis vegetative explants appearto be reliable materials for screening for genetic mutations involved inthe vegetative-to-embryonic transition. Herein we disclose theidentification of the Plant Growth Activator 6 (PGA6) gene by a novelgenetic screen. Overexpression of PGA6 promotes the formation of somaticembryos from various vegetative tissues as well as from zygotic embryosindependently of any external plant hormones. These somatic embryos,following a developmental process remarkably similar to that of zygoticembryogenesis, are able to germinate and grow into healthy, fertileplants, suggesting that PGA6 is involved in the maintenance of embryoniccell identity. PGA6 was found to be identical to the Wuschel gene (WUS),a homeodomain protein that was previously characterized as a keyregulator for specification of meristem cell fate (Laux et al., 1996;see, also, WO 01/23575 regarding WUS homologs (SEQ ID NOs:3-14)). Thenucleic acid and protein sequences of Wuschel are those as shown byGenBank Accession No. AJ012310 (SEQ ID NO:15). Our results reveal anadditional function of WUS/PGA6 during embryogenesis, and also open anew avenue in biotechnological applications.

In this study, we identified a genetic locus PGA6 by a novel functionalscreen aimed at elucidating the molecular mechanism of somaticembryogenesis. A gain-of-function mutation at this locus causes a rapidtransition from vegetative or somatic to embryonic cells, leading tosomatic embryo development from various tissues and organs. The pga6mutation-dependent cell fate reprogramming can occur either in thepresence or absence of external plant hormones, although the localconcentration of endogenous growth regulators might play an importantrole in the vegetative-to-embryonic transition. In addition, thehormone-independent somatic embryogenesis in pga6 strikingly resembleszygotic embryo development. These observations suggest that PGA6 plays acritical regulatory role during embryogenesis, likely involved inmaintaining embryonic cell identity. Molecular and genetic analysesindicate that pga6 is a gain-of-function allele of the previouslycharacterized wus loss-of-function mutation (Mayer et al., 1998).

In addition to causing vegetative tissues or organs to becomeembryogenic, inducing overexpression of PGA6 in zygotic embryos alsoresults in the generation of somatic embryos at very high frequency,whereas no somatic embryos or embryogenic calli were observed in theabsence of the inducer. Wu et al. (1992) previously reported thegeneration of somatic embryos from isolated Arabidopsis zygotic embryosat very low efficiency, which involved tedious multiple subculturing andhormone treatments. Our finding that the simple manipulation of a singlegene (WUS) was able to generate somatic embryos at very high frequencyis a major advance in plant biotechnology.

Loss-of-function mutations in WUS have been shown to cause impaireddevelopment of shoot and floral meristems in Arabidopsis, resulting inthe absence of the shoot and floral meristems in all developmentalstages of wus embryos and adult plants (Laux et al., 1996; Mayer et al.,1998). Genetic studies revealed that WUS interacts with CLAVATA (CLV),and the WUS/CLV self-regulatory loop, in which CLV presumably actsupstream of WUS (Clark, 2001) appears to be critical for the maintenanceof stem cell identity (Schoof et al., 2000; Brand et al., 2000; Waitesand Simon, 2000). On the other hand, ectopic expression of WUS resultsin enlarged meristems (Schoof et al., 2000). Collectively, theseobservations suggested an instructive role of WUS for the specificationof meristematic stem cell fate. Interestingly, the WUS gene is notexpressed in the stem or meristem cells; rather its expression isrestricted to a small group of cells underneath the stem cells duringthe entire embryogenesis and post-embryogenesis stages (Mayer et al.,1998; Schoof et al., 2000). The cluster of WUS expressing cells wastermed as the organizing center (Mayer et al., 1998). The unexpectedexpression pattern led to postulations that WUS promotes and/ormaintains the stem cell fate by a diffusion mechanism or acted in anon-cell-autonomous manner (Mayer et al., 1998; Waites and Simon, 2000).

Our observation that WUS is capable of promoting vegetative-to-embryonictransition and eventually somatic embryo formation uncovers anadditional critical function of this homeodomain protein duringembryogenesis. Presumably, the highly restrictive expression of WUShallmarks the putative embryonic organizing center which, in turn, maygive rise to stem cells during embryogenesis and later development.Therefore, WUS is involved in promoting and maintaining the identity ofembryonic cells from which stem cells are derived. BecauseWUS-expressing cells have not been morphologically and functionallycharacterized, it remains of interest to determine whether this clusterof cells indeed represents a functional organizing center similar toSpemann's organizer discovered nearly 80 years ago in Xenopus embryos(Spemann and Mangold, 1924).

Interestingly, the LEC1 transcript was barely detectable in theorganizing center or the WUS expressing domain, albeit LEC1 was found toexpress throughout embryogenesis as well as in seeds (Lotan et al.,1998). Consistent with these observations, somatic embryo expression ofLEC1, presumably resembling that in zygotic embryos, was found to bepromptly repressed by the WUS activity. In addition, the LEC1 functionappeared to require unidentified embryo- and or seed-specific cofactors,since inducible overexpression of LEC1 by the XVE system (Zuo, et al.,2000b) during post-germination stages did not result in any detectablephenotype. By contrast, WUS appears to be a key player in promotingembryonic potential as its activity does not appear to require anydevelopmentally specific factors under our tested conditions. Takentogether, these observations further suggest that WUS plays apredominant role in inducing the embryonic potential, whereas LEC1 islikely involved in promoting differentiation of embryonic cells at laterdevelopmental stages. A reasonable assumption would be that the LEC1activity, a driving force for embryo cell differentiation, must beexcluded in order to fully maintain the embryonic potential in theputative organizing center.

Recently, Stone et al. (2001) showed that LEC2 encodes a transcriptionfactor containing a B3 domain unique to several other planttranscription factors including ABI3/VP1 and FUS3. Overexpression ofLEC2 leads to formation of somatic embryos as well as the formation ofcallus, cotyledon-like and leaf-like structures, a phenotype similar tothat of pga6 mutant, suggesting that LEC2 might be functionally close toWUS. It will be interesting to determine if the LEC2 activity is alsodependent on embryo- and/or seed-specific cofactors as in the case forLEC1.

Systems for hormone-dependent somatic embryo formation have been wellestablished in several model species, and rapidly extended to otherspecies (Zimmerman, 1993; Mordhorst et al., 1997). In all these in vitrosystems, phytohormones, particularly auxin or 2,4-D, are essential forinduction of somatic embryo formation. Arabidopsis has been known to bea species difficult for somatic embryogenesis, even though with limitedsuccess by using wild-type immature zygotic embryos (e.g., Wu et al.,1992) or certain vegetative tissues of the pt mutant plants (Mordhorstet al., 1998). Overexpression of Leafy cotyledon 1 (Lec1) causes severedevelopmental abnormality and growth arrest, a phenotype similar to thatof the pga6 mutant (Lotan et al., 1998). Formation of somatic embryos isoccasionally observed in the Lec1 overexpression plants (Lotan et al.,1998), but these embryos never germinate or develop into normal adultplants. The finding that the pga6 gain-of-function mutation oroverexpression of WUS results in hormone-independent somatic embryoformation at a high frequency will have significant impact on plantbiotechnology, and provides a convenient and attractive model system formany aspects of plant biological research.

In another embodiment of the invention, embryogenesis is induced inhaploid cells, such as pollen cells, to produce haploid plants. This canbe achieved by stably transforming a plant cell or tissue with a WUSgene under the control of a tissue specific promoter that is active in ahaploid cell or tissue, and expressing the WUS gene therein, or byintroducing the WUS gene into a plant tissue or cell under the controlof an inducible promoter and applying the inducer to cause expression ofthe WUS gene therein. In a preferred embodiment, the WUS gene is underthe control of a promoter that is both haploid-tissue specific andinducible. In a preferred embodiment, a promoter is used that is bothinducible and tissue-specific, giving greater control over the process.In a most preferred embodiment, a WUS gene linked to an induciblepollen-specific promoter is used to induce somatic embryogenesis inpollen cells.

Expression of the gene in the haploid tissue or cell (for example, byapplication of the inducer specific for the inducible promoter) resultsin the formation of haploid somatic embryos, which can be grown intohaploid plants using standard techniques. When an inducible promoter isused (whether tissue specific or not), a preferred method comprisesexposing excised transgenic tissue containing the haploid cells (e.g.,pollen or ovules) to the inducer specific for the inducible promoter fora time sufficient to induce the formation of a somatic embryo,withdrawing the inducer, and growing the somatic embryo into atransgenic haploid plant in the absence of the inducer.

Diploidization of the haploid plants to form dihaploids, eitherspontaneously or by treatment with the appropriate chemical (e.g.colchicine) will significantly expedite the process of obtaininghomozygous plants as compared to a method of conventional geneticsegregation. This technology will not only be beneficial for breedingpurposes but also for basic research such as studies of mutagenesis andother genetic studies, because dihaploids are truly homozygous down tothe DNA level, containing two identical copies of each gene.

Additionally, WUS genes will be useful for inducing apomixis intoplants. Apomixis and methods of conferring apomixis into plants arediscussed in several patents (see, e.g., U.S. Pat. Nos. 5,710,367;5,811,636; 6,028,185; 6,229,064; and 6,239,327 as well as WO 00/24914which are incorporated herein by reference). Reproduction in plants isordinarily classified as sexual or asexual. The term apomixis isgenerally accepted as the replacement of sexual reproduction by variousforms of asexual reproduction (Rieger et al., IN Glossary of Geneticsand Cytogenetics, Springer-Verlag, New York, N.Y., 1976). In general theinitiation of cell proliferation in the embryo and endosperm areuncoupled from fertilization. Apomixis is a genetically controlledmethod of reproduction in plants where the embryo is formed withoutunion of an egg and a sperm. There are three basic types of apomicticreproduction: 1) apospory-embryo develops from a chromosomally unreducedegg in an embryo sac derived from a somatic cell in the nucellus, 2)diplospory-embryo develops from an unreduced egg in an embryo sacderived from the megaspore mother cell, and 3) adventitiousembryony-embryo develops directly from a somatic cell. In most forms ofapomixis, pseudogamy or fertilization of the polar nuclei to produceendosperm is necessary for seed viability. These types of apomixis haveeconomic potential because they can cause any genotype, regardless ofhow heterozygous, to breed true. It is a reproductive process thatbypasses female meiosis and syngamy to produce embryos geneticallyidentical to the maternal parent. With apomictic reproduction, progenyof specially adaptive or hybrid genotypes would maintain their geneticfidelity throughout repeated life cycles. In addition to fixing hybridvigor, apomixis can make possible commercial hybrid production in cropswhere efficient male sterility or fertility restoration systems forproducing hybrids are not known or developed. Apomixis can make hybriddevelopment more efficient. It also simplifies hybrid production andincreases genetic diversity in plant species with good male sterility.

It would be ideal to find genes controlling obligate or a high level ofapomixis in the cultivated species and be able to readily hybridizecross-compatible sexual x apomictic genotypes to produce true-breedingF₁ hybrids. In reality, most desirable genes controlling apomixis arefound in the wild species which are distantly related to the cultivatedspecies. Although interspecific crosses may be possible between thecultivated and wild species, chromosome pairing between genomes isusually low or nonexistent.

Although apomixis is effectively used in Citrus to produce uniform anddisease- and virus-free rootstock (Parlevliet et al, 1959) and inbuffelgrass (Bashaw, 1980) and Poa (Pepin et al, 1971) to produceimproved cultivars, it has not been successfully transferred to acultivated crop plant. The transfer of apomixis to important crops wouldmake possible development of true-breeding hybrids and commercialproduction of hybrids without a need for cytoplasmic-nuclear malesterility and high cost, labor-intensive production processes. Anobligately apomictic F₁ hybrid would breed true through the seedindefinitely and could be considered a vegetative or clonal method ofreproduction through the seed. The development of apomicticallyreproducing cultivated crops would also provide a major contributiontoward the food security in developing nations (Wilson et al, 1992).

The present invention is further detailed in the following Examples,which are offered by way of illustration and are not intended to limitthe invention in any manner. Standard techniques well known in the artor the techniques specifically described below are utilized.

Example 1 Plant Materials, Growth Conditions and Plant Transformation

The Wassilewskija, Columbia and Landsberg ecotypes of A. thaliana wereused. Plants were grown under continuous white light at 22° C. on solidA medium (1×MS salts (Murashige and Skoog, 1962), 3% sucrose, 0.8% agar)supplemented with appropriate antibiotics and/or the inducer17-β-estradiol. Unless indicated otherwise, 5 μM 17-β-estradiol was usedfor induction. In planta transformation of Arabidopsis plants (theColumbia ecotype) was performed as described by Bechtold et al. (1993).Transformation of root explants was carried out according to Koncz etal. (1989).

Light and electron microscope analyses were carried out as previouslydescribed (Zuo et al., 2000b).

Example 2 Screening of pga Mutants

Agrobacteria ABI cells carrying pER16 were used to transform Arabidopsis(the Wassilewskija ecotype) root explants. Infected root explants werecultured on the screening medium (SCM; 1×MS salts, 1% sucrose, 0.5 g/LMES (2-[N-morpholino]ethanesulfonic acid), 0.15 mg/L IAA (indole aceticacid), 5 μM 17-β-estradiol, 50 mg/L kanamycin, 100 mg/L carbenicillinand 0.2% phytagel, pH 5.7) at 22° C. under a 16-hour white light/8-hourdark cycle. Putative pga mutants, which appeared as rapidly growinggreen-yellowish or green cell clumps or calli upon culturing on the SCMfor 10-15 days, were transferred onto a non-inductive shoot inductionmedium (SIM; for green calli; 1×MS salts, 1% sucrose, 0.5 g/L MES, 1mg/L 2-IP (N⁶, δ²-isopentenyladenine), 0.15 mg/L IAA, 50 mg/L kanamycin,100 mg/L carbenicillin and 0.2% phytagel, pH 5.7) to recover mutantshoots. The green-yellowish calli were transferred onto the callusinduction medium (CIM; 1×B5 salts (Sigma), 2% glucose, 0.5 g/L MES, 0.5mg/L 2,4-D, 0.05 mg/L kinetin, 50 mg/L kanamycin, 100 mg/L carbenicillinand 0.2% phytagel, pH 5.7). After culturing on the CIM for 7-10 days,the amplified calli were then transferred onto a SIM to regenerateshoots. Regenerated shoots, usually formed after culturing on the SIMfor 2-3 weeks, were then transferred to a root induction medium (RIM;identical to SIM but without 2-IP) to promote root formation. Whereasmost putative pga mutant plantlets, including pga6, were able to setseeds after transferring to soil, a portion of roots were excised andplaced on the CIM to reinduce callus formation, followed by repeatingthe above-described screening procedure to confirm the inducer-dependentpga phenotype. The pga6 mutant was backcrossed with wild-type(Wassilewskija) plants twice for further genetic and phenotypicanalyses.

Example 3 Molecular Manipulations

Molecular manipulations were performed as specifically stated or by themethods as taught by Sambrook et al. (1989). The XVE inducibleexpression vector pER10 is identical to pER8 (Zuo et al., 2000a) exceptthat the hygromycin selectable marker of pER8 was replaced with akanamycin selectable marker. To construct the mutagenesis vector pER16,pER10 was digested with SpeI and Asp7181 followed by Klenow enzymefill-in reaction and religation. The resulting pER16 vector lacked therbcsS3A polyA addition sequence of the O^(LexA-46)::T^(3A) expressioncassette (see FIG. 1 of Zuo et al. (2000a)).

pER16 is shown in FIG. 1. Only the region between the Right Border (RB)and Left Border (LB) is shown (not to scale). Two transcription unitsand the O^(LexA)-46 promoter are located between the RB and LB. In thefirst transcription unit, the G10-90 promoter (Ishige et al., 1999)drives the XVE fusion gene terminated by the rbcs E9 polyA additionsequence. The second transcription unit consists of the NopalineSynthase (NOS) gene promoter, the coding sequence of the NeomycinPhosphotransferase II (NPT II) gene and the NOS polyadenylationsequence. The O^(LexA)-46 promoter consists of 8 copies of the LexAoperator sequence fused to the -46 CaMV35S promoter. Upon integrationinto the plant genome, the O^(LexA)-46 promoter can activate thetranscription of sequences fused downstream from the promoter in a17-β-estradiol-dependent fashion.

The pER16-tagged genomic sequences were recovered by Tail-PCR (Liu etal., 1995), and the purified PCR fragments were directly subjected toDNA sequencing analysis.

The WUS cDNA was amplified from flower cDNA by polymerase chain reaction(PCR), using the primers WusUp (5′ CTTATTTACCGTTAACTTGTGAACA 3′) (SEQ IDNO:1) and WusLow (5′ CACATAACGAGAGATAACTAGTTAAC 3′) (SEQ ID NO:2). The1062-bp long PCR fragment, harboring the entire protein coding regionplus part of the 3′ UTR, was first cloned into the pPCR-Script Amp SK(+)vector (Stratagene) and then cut with XhoI (vector polylinker site) andSpeI (site in the 3′ UTR of the WUS cDNA) for the subsequent cloninginto the corresponding sites of both the pER10 vector (17-β-estradiolinducible) and the pBA002 vector (constitutive 35S promoter) for the35S::WUS expression. The correct sequence of the WUS cDNA was confirmedby DNA sequencing analysis.

Genomic DNA Southern and RNA Northern blotting analyses were carried outas previously described (Zuo et al., 2000a; Zuo et al., 2001).

Example 4 Screening of the Plant Growth Activator Mutants

Explants derived from Arabidopsis vegetative tissues are known to beincapable of forming somatic embryos or embryogenic calli promoted byexternal plant hormones. We presumed that external hormones alone wereincapable of activating key regulators of Arabidopsis necessary forvegetative-to-embryogenic transition. With appropriate hormonetreatments, gain-of-function mutations in these regulatory genes mayactivate the vegetative-to-embryonic transition. Such gain-of-functionmutations, on the other hand, may also cause severe defects duringsubsequent plant growth and development. Therefore, if the expression ofthe mutated gene and/or the biological activity of related gene productsis not appropriately controlled, it will be difficult to maintain theidentified mutations. An example is the constitutive overexpression ofthe LEC1 gene (Lotan et al., 1998). As a consequence, we carried out thescreen by using a previously developed chemical-inducible XVE system,which has been demonstrated to be stringently controlled and to behighly responsive to the inducer 17-β-estradiol, a mammalian hormonewith no apparent physiological effects on plant growth and development(Zuo et al., 2000a). The use of an inducible promoter thus allows us torecover normal mutant plants by withdrawal of the inducer, even in thecase that the gain-of-function mutation is lethal.

Arabidopsis root explants were transformed with Agrobacteria (Koncz etal., 1989) carrying an XVE vector pER16 (FIG. 1). Transformed explantswere cultured on a screening medium (SCM containing auxin,17-β-estradiol and kanamycin but without cytokinin). Note that mutationsfunctionally analogous to cytokinin independent 1 (cki1; Kakimoto, 1996)should also be recovered under the screening conditions. In the primaryscreen, we isolated 35 putative mutants by interrogating approximately38,000 independent transformation events. As expected, most of thesemutants (33 out of 35) showed a cki1-like phenotype, i.e., shootregeneration independent of cytokinin. The remaining two mutants gaverise to green-yellowish embryogenic calli. We collectively named thesetwo classes of mutations as pga for plant growth activator. Here, wedisclose a detailed characterization of one of these mutants which isnamed pga6.

Example 5 The pga6 Gain-of-Function Mutation Promotes theVegetative-to-Embryonic Transition

The pga6 mutant was initially identified by its ability to formembryogenic calli on SCM. The embryogenic calli were transferred onto ashoot induction medium containing both auxin and cytokinin but withoutthe chemical inducer 17-β-estradiol. After 2-3 weeks, shoots wereregenerated from the isolated calli. Explants derived from differentorgans of the regenerated shoots were cultured on SCM as describedbefore; a portion of the excised explants was cultured in SCM withoutthe inducer to serve as controls. After culturing for 7-10 days, onlyslowly growing calli were occasionally observed in the absence ofinducer (FIG. 2A). In the presence of the inducer, however, the explantsproduced numerous, rapidly growing, yellowish embryogenic calli (FIG.2B), which subsequently developed into distinctive somatic embryos (FIG.2C). After being transferred onto a non-inductive medium, all thesesomatic embryos were able to germinate (FIGS. 2D and 2E) and developinto fertile adult plants, most of which were morphologicallyindistinguishable from wild-type plants (FIG. 2F).

To further confirm the above observations, T1 seeds collected from theT0 somatic embryo-derived plants, as shown in FIG. 2F, were germinatedon MS medium in the absence of the inducer. The inducer-dependentsomatic embryo formation was reproducibly observed in pga6 explantsprepared from different organs/tissues of previously uninduced T1plants. Similar to that observed in the T0 explants, the highestfrequency of somatic embryo formation was observed from root explants,followed by leaf petioles, stems and leaves. Isolated zygotic embryoshad a frequency similar to that of root explants. The above resultssuggested that the pga6 gain-of-function mutation was able to promotevegetative-to-embryonic transition under our experimental conditions,and that the PGA6 gene was most likely tagged by the inducer-responsiveO^(LexA)-46 promoter.

In addition to the formation of somatic embryos, which are characterizedby the presence of cotyledons lacking trichomes on the surfaces, we alsoobserved approximately 10% of the pga6 calli developing into shoots,suggesting that PGA6 is also involved in organogenesis.

Example 6 The Development of pga6 Somatic Embryos Morphologically andTemporally Resembles that of Zygotic Embryos

To more closely follow the somatic embryogenesis process of the pga6mutant, we performed a scanning electron microscopic analysis. Somaticembryogenesis in the inducer-treated pga6 explants highly resembledzygotic embryogenesis. The process was initiated from an asymmetricdivision of a single cell, giving rise to a smaller apical cell and alarger basal cell (FIG. 3A). Subsequently, embryo-like structuresequivalent to that of zygotic embryos at the early globular stage wereeasily recognizable (FIG. 3B). These somatic embryos underwent a typicalembryogenesis process, including the heart stage, the torpedo stage, andthe cotyledon stage (FIGS. 3B and 3C), and eventually germinated intohealthy seedlings (FIG. 2F). In addition to the relatively normal growthand development of the somatic embryo-derived mutant plants, we observedthat a small fraction (approximately 10%) of somatic embryos generatedseedlings with three cotyledons (FIG. 3D). This abnormality ispresumably caused by different expression levels of the PGA6 gene or byabiotic effects as occasionally observed in seedlings germinated fromwild-type seeds. Nevertheless, these above results demonstrated that aconditional gain-of-function mutation in the pga6 locus promotedvegetative-to-embryonic transition, leading to the initiation of somaticembryogenesis.

Example 7 Plant Hormone-Independent Somatic Embryo Formation

The data described above were obtained under tissue culture conditions.To investigate the effects of the pga6 mutation on normal plant growthand development, T1 seeds were germinated on MS media (Murashige andSkoog, 1962) with or without the inducer. No detectable abnormality ingrowth and development (phenotype) was observed in pga6 plantsgerminated and grown in the absence of the inducer. On the inductive MSmedium, progeny with the mutant and wild-type phenotype segregated inthe ratio of 3:1, characteristic of a dominant mutation in a singlelocus. Compared to wild-type seeds, the pga6 mutant seeds germinatedsubstantially later, with a delay of 3-5 days, suggesting an inhibitoryeffect of the mutation on plant growth and development. Whereasapproximately one third of the mutant seedlings stopped further growthafter germination and eventually died, the remaining two thirds mutantseedlings rapidly turned into green calli upon germination. These twodistinctive mutant phenotypes, with an approximately 2:1 ratio, werepresumably due to segregations (heterozygous or homozygous) for the pga6gain-of-function mutation locus, leading to different PGA6 expressionlevels in homozygous and heterozygous seedlings. This notion wassupported by subsequent genetic analysis of the T2 generation derivedfrom T1 plants grown under non-inductive conditions. Whereas allprogenies of the putative homozygous T2 families (5 out of 16) showedonly the “lethal” phenotype on the inductive medium, the remaining 11heterozygous families gave rise to a typical 2:1:1 segregation forembryogenic calli, “lethal” and wild-type phenotypes. In addition, thestrength of the mutant phenotype was dependent on the inducerconcentrations and the induced PGA6 expression levels per se (see FIGS.5B-C).

In the absence of any external plant hormones, somatic embryos wereformed from green embryogenic calli (FIGS. 4B and 4C), which were ableto germinate and grow normally into fertile adult plants (FIG. 4D).Interestingly, a considerably higher frequency of embryogenic callusformation was observed from excised pga6 roots cultured on the inductiveMS medium (FIGS. 4G and 4H), suggesting the presence of an antagonisticactivity to PGA6 in intact plants, which may promote post-embryogenesisgrowth and development.

Overexpression of LEC1 leads to abnormal plant growth and development aswell as the occasional formation of embryo-like structures (Lotan etal., 1998). The LEC1 gain-of-function phenotype, however, appeared to bestrictly restricted to developmental stages prior to germination. Afterseedling germination, overexpression of LEC1, controlled by the XVEinducible expression system (Zuo et al., 2000a), did not produce anyapparent abnormality in plant growth and development, although the LEC1transgene was highly responsive to the inducer during post-germinationstages. On the other hand, the same transgenic line showed a strongphenotype if germinated directly in the presence of the inducer and theLEC1 transgene was highly responsive to the inducer duringpost-germination stages. These observations suggest that embryo- orseed-specific co-factor(s) are required for LEC1 function. To examine ifPGA6 function is also dependent on embryo- or seed-specific co-factors,pga6 mutant seedlings at different growth stages, germinated and grownon the non-inductive MS medium, were transferred onto an inductive MSmedium. At these developmental stages, the fate of both root and shootstem cells has already been highly specified in wild-type plants. Thepga6 mutation, however, appeared to reverse the developmental program,causing both root and shoot meristems to transform into embryogeniccalli (FIG. 4D). Similar to that shown before, these embryogenic calliwere eventually capable of forming somatic embryos, which were able togerminate and grow into morphologically normal adult plants. The aboveresults strongly suggest that PGA6 plays a key role in specifying andmaintaining embryonic cell identity, independent of any embryo- orseed-specific co-factors.

Example 8 PGA6 is Identical to the Homeodomain Protein WUS

Based on the segregation of the kanamycin selectable marker, the pga6mutant genome appears to contain a single transgenic locus. However,molecular analysis indicated the presence of the O^(LexA)-46 promoter intwo independent loci. One O^(LexA)-46 promoter was found to fuse to theWUS gene in chromosome II (Mayer et al., 1998), approximately 1kilobase-pair (Kb) upstream from the putative translation initiationcodon (FIG. 5A). The second O^(LexA)-46 promoter fused immediatelyupstream of the putative translation initiation codon of an open readingframe (ORF) in chromosome V, encoding a putative basic-helix-loop-helixtype transcription factor (deigned ORF1). To verify the identity of thePGA6 gene(s), cDNA fragments containing both WUS and the putative ORF1were cloned into an XVE vector, and the resulting constructs were usedto transform wild-type plants (Bechtold et al., 1993). Explants derivedfrom XVE-ORF1 T1 transgenic plants did not show any apparentinducer-dependent phenotype. In addition, ORF1 expression did not appearto be up-regulated by the chemical inducer in pga6 plants, presumablydue to the instability of the ORF1 transcript lacking the entire5′-untranslated region (UTR). By contrast, all pga6 mutant phenotypes asdescribed before were observed in the XVE-WUS T2 transgenic plants(FIGS. 6A and 6B) in an inducer-dependent manner (see Example 11 fordetails).

WUS expression was strictly dependent on different concentrations of theinducer in pga6 mutant plants (FIG. 5B). FIG. 5C shows expression ofPGA6/WUS induced by different concentrations of the inducer. Ten-day-oldpga6 seedlings germinated and grown on the MS medium were transferred toan MS medium containing various concentrations of 17-β-estradiol asindicated and cultured for an additional 16 hours before total RNAextraction. Five μg total RNA were used for Northern blot analysis usinga WUS cDNA fragment as a probe. Consistent with the inducerconcentration-dependent WUS expression, pga6 plants also showed variouspenetrations of the mutant phenotype in an inducerconcentration-dependent fashion (FIG. 5B), thus providing a series ofconditional mutant alleles for further functional studies.

We also used a 35S-WUS construct for transformation of Arabidopsisthaliana Columbia as well as Landsberg ecotype. Most of thetransformants recovered from selection showed strong alteration inseedling development. The hypocotyl was swollen and root tips oftenenlarged to give rise to shoot-like or embryo-like structures which wereunable to further develop (FIGS. 6C-G). Leaf development, when observed,was also compromised indicating strong alteration of the shoot apicalmeristem (SAM) which, besides lateral organs, could also formadventitious shoots or embryo-like structures (FIGS. 6G-H). Overall, allthe tissues of the transformants would give rise to organs orembryo-like structures whose further development, however, was impaired.This observation supports the validity of our strategy of using aninducible system to isolate genes involved in the switch to embryodevelopment as the continuous overexpression of regulatory proteinswould prevent recovery of the mutants.

In summary, the above results indicated that the O^(LexA)-46promoter-tagged WUS in the mutant genome represents PGA6. We will referto the PGA6 gene/protein as WUS in the future, but use pga6 for themutation/mutant identified in this study due to different propertiesbetween the loss- and gain-of-function mutations.

Because the O^(LexA)-46 promoter fused approximately 1 Kb upstream fromthe putative translation start codon (FIG. 5A), the WUS promoterpresumably remains functional in the mutant genome, leading to noapparent loss-of-function phenotype for the mutation. Nevertheless, theWUS gene was strongly inducible, giving rise to two transcripts,approximately 1.3 and 2.3 Kb (FIG. 5C). The shorter transcript waspresumably generated from the native transcription initiation site ofthe WUS gene, in which case the LexA operator sequence might serve as anenhancer to the WUS promoter. On the other hand, the longer transcriptmight represent transcription from the O^(LexA)-46 promoter. Thissuggests that the O^(LexA)-46 sequence can serve as a functionalpromoter, as well as a transcriptional enhancer for activation tagging.

Example 9 WUS Represses LEC1 Expression During Embryogenesis

The above data indicate that WUS, in addition to its meristem functiondescribed previously (Laux et al., 1996; Mayer et al., 1998), also playsa critical role in promoting or maintaining the embryonic potential. Wehave investigated expression of several embryo- or seed-specific genesin pga6 embryogenic callus and somatic embryos. Root explants derivedfrom pga6 mutant plants were cultured on an MS medium supplemented withthe inducer. Under such conditions, whereas embryogenic calli appearedafter 10-15 days, somatic embryos and germinating seedlings weregenerated after 20 days (see FIGS. 2A-F, 3A-D and 4A-H). Due to thetransient expression nature of the XVE inducible system (Zuo et al.,2000a), WUS expression gradually declined upon continued culture on theinductive medium; however, the expression can be strongly re-induced byadding freshly-prepared inducer (FIG. 7A).

The LEC1 gene, normally expressed only in embryos and seeds (Lotan etal., 1998), was highly expressed in 20-30-day-old explants, a stage whensomatic embryos and derived seedlings were generated. FIG. 7B shows LEC1expression from 14 days until 28 days when pga6-dependent somaticembryogenesis takes places. LEC1 expression, however, was dramaticallydecreased upon reactivation of WUS expression (FIG. 7B). No alterationof LEC1 expression was detected when the explants/calli were transferredonto the control medium for an additional two days, suggesting that theLEC1 repression was a specific response to the 17-β-estradiol inducedWUS expression.

These observations suggest that LEC1 expression in pga6 explants was nota direct response to WUS overexpression but rather a consequence of thepga6 somatic embryo development. On the other hand, a developmental pathredefined by WUS overexpression leads to the repression of LEC1, a genepresumably involved in embryo maturation.

Example 10 Enhanced Frequency of Somatic Embryo Formation by Addition of2,4-D

Although we were able to generate somatic embryos from various pga6tissues/organs in the absence of any external hormone, the frequency ofsomatic embryo formation appears to be lower compared to that observedin our original screening conditions, under which a 2,4-D pretreatmentwas included. To test the effects of 2,4-D on somatic embryogenesis,pga6 root explants were cultured on MS medium with 0.5 mg/L 2,4-D for 5days prior to being transferred to an MS medium with or without 10 μM17-β-estradiol. No somatic embryo formation was observed in the mediumwithout the inducer, whereas numerous somatic embryos were generatedafter 2-3 weeks culturing in the presence of the inducer. As shown above(see Example 7 and FIGS. 4A-H) and by the results disclosed in thisExample, the absence of 2,4-D pretreatment resulted in a substantiallydecreased efficiency for somatic embryo formation, indicating that the2,4-D treatment is able to significantly increase the efficiency ofsomatic embryo formation. Collectively, the above results suggest that2,4-D was able to enhance yet unidentified components in the signalingnetwork, which play a key role in promoting the vegetative-to-embryonictransition. In addition, these results further demonstrated the validityof our working hypothesis as well as the screening strategy, i.e., thatexternal hormones alone were incapable of activating key components forthe vegetative-to-embryonic transition in Arabidopsis, and that theappropriate external hormone treatment in combination withgain-of-function mutations in key regulatory genes is fully capable ofpromoting the vegetative-to-embryonic transition or somaticembryogenesis.

Example 11 Somatic Embryo Formation of Explants Derived from TransgenicPlants Carrying a pER10-WUS Transgene

The XVE vector pER10 is identical to pER8 except that the hygromycinresistance marker is replaced with a kanamycin-resistance marker (Zuo etal., 2000(a)). Full length WUS cDNA was placed under the control of theXVE system in pER10. Stem segments derived from the pER10-WUS transgenicplants were pre-cultured on the MS medium with 0.5 mg/L 2,4-D for 5 daysand then transferred to an MS medium with or without 10 μM17-β-estradiol. No somatic embryo was observed in medium in the absenceof the inducer, whereas with the inducer, many somatic embryos weregenerated after 2-3 weeks of culture.

Similar to that of pga6 mutant plants, we found that the 2,4-Dpre-treatment substantially increased the frequency of somatic embryoformation in pER10-WUS transgenic plants.

The method of placing WUS in pER10 or similar vectors which can beregulated enables one to perform the same or similar experiments inplants other than Arabidopsis.

Example 12 Placing WUS Under the Control of a Tissue Specific Promoter

The G10-90 promoter in the XVE vector can be replaced with atissue-specific promoter (e.g. a pollen-, root-stem- or leaf-specificpromoter). A variety of tissue specific promoters are well known tothose of skill in the art. Because expression of a transgene isactivated by the chimeric XVE gene which is controlled by atissue-specific promoter in this Example, the O^(LexA)-46 promotercontrolling the WUS transgene is therefore tissue-specific in aninducer-dependent manner. This means that WUS will be induced only inthe presence of an inducer and only in the specific tissue correspondingto the tissue specific promoter. Appropriate tissues or cell types, canthen be collected from the transgenic plants and used for induction ofsomatic embryos as described in Examples 10 and 11.

Particularly when pollen derived from transgenic plants carrying apollen-specific promoter-XVE/O^(LexA)-46-WUS vector is used, progenyplants generated from pollen-derived somatic embryos should be haploidinstead of diploid (see, e.g., Twell et al., 1989 and Twell et al., 1990for pollen specific promoters). In this embodiment of the invention, atransgenic plant having in its genome a Wuschel (WUS) gene under thecontrol of an inducible, pollen-specific promoter would not normallyexpress the gene. Pollen from such a plant can be cultured in thepresence of the inducer until somatic embryogenesis occurs, after whichthe inducer is removed and the haploid embryos are permitted to developinto haploid clones according to standard techniques.

Example 13 Use of the pER10-WUS as a Silent Marker for Transformation

The pER10-WUS vector can be used directly for transformation of explantswithout the use of an antibiotic resistance marker. Somatic embryos thatformed in the presence of an inducer but in the absence of cytokininshould be transformants, because under such conditions non-transformantswill be incapable of forming somatic embryos nor shoots due to the lackof induced WUS gene expression. Upon inducer removal, the embryos andshoots will develop into normal and fertile plants. The vector caninclude any gene or genes which are desired to be present in thetransformed plants and these can be under the control of a desiredpromoter. The plants selected as a result of selecting for inducible WUSexpression-dependent somatic embryos or shoots will contain the desiredgene or genes.

If desired, the WUS transgene can be placed into a vector comprising ameans of removing the WUS transgene as well as other portions of thevector which are no longer desired, e.g., using the XVE-Cre/lox system(Zuo et al., 2001). Such methods are also disclosed in U.S. patentapplication Ser. No. 09/439,534, filed 12 Nov. 1999, which isincorporated herein by reference.

Example 14 Induction of Formation of Somatic Embryos from IsolatedZygotic Embryos of PGA6 Transgenic Plants

Zygote embryos at late heart stage were isolated from young siliques ofPGA6 plants and transferred to either non-inductive medium (MICK: MSsalts, 30 g/L sucrose, 0.15 mg/L IAA, 100 mg/L carbenicillin, 50 mg/Lkanamycin, and 0.25% phytagel, pH 5.7) or inductive medium (MICK plus10.0 μM 17-β-estradiol). After two weeks of culture, 10 zygotic embryosformed embryogenic calli on the inductive medium, but only 3 zygoticembryos started to form embryogenic calli on the non-inductive MICKmedium. After 35 days of culture on the inductive medium, 10 zygoticembryos formed somatic embryos with a regeneration index (the number ofsomatic embryos per zygote embryo explant) of about 70-100 (FIG. 8A). Bycontrast, on MICK medium without estradiol, only two zygotic embryosregenerated somatic embryos and the somatic embryo regeneration indexwas about 10-20 (FIG. 8B).

Example 15 Generating an Apomictic Plant

Apomixis can be induced by introducing WUS into a plant cell in such amanner that the WUS gene is expressed in the appropriate tissues (e.g.,nucellus tissue). This can be by means of, but is not limited to,placing the WUS gene under the control of a tissue-specific promoter(e.g., a nucellus-specific promoter), an inducible promoter, or apromoter that is both inducible and tissue-specific. Inducing expressionof the WUS gene, e.g. in the nucellus, produces apomixis leading to anapomictic plant. This plant may then be used to establish atrue-breeding plant line. Additionally, the vector utilized to transferWUS into the plant cell can include any other desired heterologous genein addition to WUS, including but not limited to, a marker gene or agene to confer a desirable trait upon the plant, e.g., a gene resultingin larger plants, faster growth, resistance to stress, etc. This wouldlead to the development of an apomictic line with the desired trait.

In a variation of the scheme, plant expression cassettes, including butnot limited to monocot or dicot expression cassettes, directing WUSexpression to the inner integument or nucellus can easily beconstructed. An expression cassette directing expression of the WUS DNAsequences to the nucellus is made using the barley Nuc1 promoter (Doanet al., 1996). The expression is used for plant transformation. Othergenes which confer desirable traits can also be included in thecassette.

It is anticipated that transgenic plants carrying the expressioncassette will then be capable of producing de novo embryos from WUSexpressing nucellar cells. In the case of maize, this is complemented bypollinating the ears to promote normal central cell fertilization andendosperm development. In another variation of this scheme, Nuc1:WUStransformations could be done using a fie (fertility-independentendosperm)-null genetic background which would promote both de novoembryo development and endosperm development without fertilization (Ohadet al., 1999). Upon microscopic examination of the developing embryos itwill be apparent that apomixis has occurred by the presence of embryosbudding off the nucellus. In yet another variation of this scheme theWUS DNA sequences could be delivered as described above into ahomozygous zygotic-embryo-lethal genotype. Only the adventive embryosproduced from somatic nucellus tissue would develop in the seed.

While the invention has been disclosed by reference to the details ofpreferred embodiments of the invention, it is to be understood that thedisclosure is intended in an illustrative rather than in a limitingsense, as it is contemplated that modifications will readily occur tothose skilled in the art, within the spirit of the invention and thescope of the appended claims.

LIST OF REFERENCES

-   Bashaw, Crop Science, 1980. 20: p. 112.-   Bechtold, N., Ellis, J., and Pelletier, G., In planta    Agrobacterium-mediated gene transfer by infiltration of adult    Arabidopsis thaliana plants. C. R. Acad. Sci. Ser. III Sci.    Vie, 1993. 316: p. 1194-1199.-   Brand, U., Fletcher, J. C., Hobe, M., Meyerowitz, E. M., and Simon,    R., Dependence of stem cell fate in Arabidopsis on a feedback loop    regulated by CLV3 activity. Science, 2000. 289: p. 617-619.-   Clark, S. E., Cell signalling at the shoot meristem. Nat. Rev. Mol.    Cell Biol., 2001. 2: p. 276-284.-   Doan, D. N., Linnestad, C. and Olsen, O. A., Isolation of molecular    markers from the barley endosperm coenocyte and the surrounding    nucellus cell layers. Plant Mo. Biol., 1996. 31: p. 877-886.-   Ishige, F., Takaichi, M., Foster, R., Chua, N.-H., and Oeda, K., A    G-box motif (GCCACGTGCC) tetramer confers high-level constitutive    expression in dicot and monocot plants. Plant J., 1999. 18:443-448.-   Kakimoto, T., CKI1, a histidine kinase homolog implicated in    cytokinin signal transduction. Science, 1996. 274: p. 982-985.-   Koncz, C., Martini, N., Mayerhofer, R., Koncz-Kalman, Z., Korber,    H., Redei, G. P., and Schell, J., High frequency T-DNA-mediated gene    tagging in plants. Proc. Natl. Acad. Sci. USA, 1989. 86(21): p.    8467-8471.-   Laux, T., Mayer, K. F., Berger, J., and Jurgens, G., The WUSCHEL    gene is required for shoot and floral meristem integrity in    Arabidopsis. Development, 1996. 122: p. 87-96.-   Lin, X., Hwang, G. J, and Zimmerman, J. L., Isolation and    characterization of a diverse set of genes from carrot somatic    embryos. Plant Physiol., 1996. 112: p. 1365-1374.-   Liu, Y. G., Mitsukawa, N., Oosumi, T., and Whittier, R. F.,    Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert    junctions by thermal asymmetric interlaced PCR. Plant J., 1995.    8: p. 457-463.-   Lotan, T., Ohto, M., Yee, K. M., West, M. A., Lo, R., Kwong, R. W.,    Yamagishi, K., Fischer, R. L., Goldberg, R. B., and Harada, J. J.,    Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo    development in vegetative cells. Cell, 1998. 93: p. 1195-1205.-   Mayer, K. F., Schoof, H., Haecker, A., Lenhard, M., Jurgens, G.,    Laux, T., Role of WUSCHEL in regulating stem cell fate in the    Arabidopsis shoot meristem. Cell, 1998. 95: p. 805-815.-   Meinke, D. W., A homoeotic mutant of Arabidopsis thaliana with leafy    cotyledons. Science, 1992. 258: p. 1647-1650.-   Meinke, D. W., Franzmann, L. H., Nickle, T. C., and Yeung, E. C.,    Leafy cotyledon mutants of Arabidopsis. Plant Cell, 1994. 6: p.    1049-1064.-   Mordhorst, A. P., Toonen, M. A. J., and de Vries, S. C., Plant    embryogenesis. Crit. Rev. Plant Sci., 1997. 16: p. 535-576.-   Mordhorst, A. P., Voerman, K. J., Hartog, M. V., Meijer, E. A., van    Went, J., Koornneef, M., and de Vries, S. C., Somatic embryogenesis    in Arabidopsis thaliana is facilitated by mutations in genes    repressing meristematic cell divisions. Genetics, 1998. 149: p.    549-563.-   Murashige, T., and Skoog, F., A revised medium for rapid growth and    bioassays with tobacco tissue culture. Physiol. Plant, 1962. 15: p.    473-497.-   Ohad, N., Yadegari, R., Margossian, L., Hannon, M., Michaeli, D.,    Harada, J. J., Goldberg, R. B., and Fischer, R. L., Mutations in    FIE, a WD polycomb group gene, allow endosperm development without    fertilization. The Plant Cell, 1999. 11: p. 407-416.-   Parlevliet J E et al. Citrus. Proc. Am. Soc. Hort. Sci., 1959.    74: p. 252-260.-   Pepin et al, Crop Science, 1971. 11: p. 445-448.-   Rieger et al., in Glossary of Genetics and Cytogenetics,    Springer-Verlag, New York, N.Y., 1976-   Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular cloning: A    Laboratory Manual. 1989, Cold Spring Harbor, N.Y.: Cold Spring    Harbor Laboratory Press.-   Schmidt, E. D., Guzzo, F., Toonen, M. A., and de Vries, S. C., A    leucine-rich repeat containing receptor-like kinase marks somatic    plant cells competent to form embryos. Development, 1997. 124: p.    2049-2062.-   Schoof, H., Lenhard, M., Haecker, A., Mayer, K. F., Jurgens, G., and    Laux, T., The stem cell population of Arabidopsis shoot meristems in    maintained by a regulatory loop between the CLAVATA and WUSCHEL    genes. Cell, 2000. 100: p. 635-644.-   Skoog, F., and Miller, C. O., Chemical regulation of growth and    organ formation in plant tissues cultured in vitro. Symp. Soc. Exp.    Biol., 1957. 11: p. 118-131.-   Spemann, H., and Mangold, H., Uber Induktion von Embryonalanlagen    durch Implantation artfremder Organisatoren. Archiv fur    Mikroskopische Anatomie and Entwicklungsmechanik, 1924. 100: p.    599-638.-   Stone, S. L., Kwong, L. W. Yee, K. M., Pelletier, J., Lepiniec, L.,    Fisher, R. L., Goldberg, R. B., and Harada, J. J., LEAFY COTYLEDON 2    encodes a B3 domain transcription factor that induces embryo    development. Proc. Natl. Acad. Sci. U.S.A., 2001. 98:11806-11811.-   Sugiyama, M., Organogenesis in vitro. Curr. Opin. Plant Biol., 1999.    2: p. 61-64.-   Sugiyama, M., Genetic analysis of plant morphogenesis in vitro. Int.    Rev. Cytol., 2000. 196: p. 67-84.-   Thomas, T. L., Gene expression during plant embryogenesis and    germination: an overview. Plant Cell, 1993. 5: p. 1401-1410.-   Twell, D., Wing, R. Yamaguchi, J. and McCormick S. Isolation and    expression of an anther-specific gene from tomato. Mol. Gen.    Genet., 1989. 217: p. 240-245.-   Twell, D. Yamaguchi, J. and McCormick, S. Pollen-specific gene    expression in transgenic plants: coordinate regulation of two    different tomato gene promoters during microsporogenesis.    Development, 1990. 109: p. 705-713.-   Waites, R., and Simon, R., Signaling cell fate in plant meristems:    Three clubs on one tousle. Cell, 2000. 103: p. 835-838.-   Wilson et al, Proceedings of the International Workshop on Apomixis    in Rice, Changsha, People's Republic of China, Jan. 13-Jan.    15, 1992. Hunan Hybrid Rice Research Center, Changsha, People's    Republic of China.-   Wu, Y., Haberland, G., Zhou, C., and Koop, H.-U., Somatic    embryogenesis, formation of morphogenetic callus and normal    development in zygotic embryos of Arabidopsis thaliana in vitro.    Protoplasma, 1992. 169: p. 89-96.-   Zimmerman, J. L., Somatic embryogenesis: A model for early    development in higher plants. Plant Cell, 1993. 5: p. 1411-1423.-   Zuo, J., Niu, Q.-W., and Chua, N.-H., An estrogen receptor-based    transactivator XVE mediates highly inducible gene expression in    transgenic plants. Plant J., 2000a. 24: p. 265-273.-   Zuo, J., Niu, Q.-W., Nishizawa, N., Wu, Y., Kost, B., and Chua,    N.-H., KORRIGAN, an Arabidopsis endo-1,4-b-glucanase, localizes to    the cell plate by polarized targeting and is essential for    cytokinesis. Plant Cell, 2000b. 12(7): p. 1137-1152.-   Zuo, J., Niu, Q.-W, Moller, S. G., and Chua, N.-H.,    Chemical-regulated, site-specific DNA excision in transgenic plants.    Nat. Biotechnol., 2001. 19. p. 157-161.

PATENTS AND PATENT APPLICATIONS

-   Published Patent Application WO 00/24914, published 4 May 2000.-   Published Patent Application WO 01/23575, published Apr. 5, 2001.-   U.S. Pat. No. 5,710,367-   U.S. Pat. No. 5,811,636-   U.S. Pat. No. 6,028,185-   U.S. Pat. No. 6,229,064-   U.S. Pat. No. 6,239,327

1. A method for producing a haploid plant comprising (a) stablytransforming a plant cell with a DNA molecule comprising at least oneWuschel coding sequence under the control of a promoter to produce atransgenic plant cell, wherein the promoter is selected from the groupconsisting of a haploid tissue specific promoter, an inducible promoterand a promoter that is both haploid-tissue specific and inducible,wherein the Wuschel coding sequence is the coding sequence of SEQ IDNO:15; (b) generating a transgenic plant from said transgenic plantcell, (c) overexpressing the Wuschel coding sequence in a haploid tissueof said transgenic plant to produce a haploid somatic embryo, (d)growing said embryo into a haploid plant.
 2. The method of claim 1,wherein the promoter is a haploid tissue specific promoter.
 3. Themethod of claim 2, wherein the promoter is a pollen-specific promoter,and the haploid tissue of the transgenic plant is pollen.
 4. The methodof claim 2, wherein the promoter is an ovule-specific promoter, and thehaploid tissue of the transgenic plant is ovule tissue.
 5. The method ofclaim 1, wherein the promoter is an inducible promoter.
 6. The method ofclaim 1, wherein the promoter is both haploid-tissue specific andinducible.
 7. The method of claim 6, wherein the promoter ispollen-specific, and the haploid plant cell is a pollen cell.
 8. Themethod of claim 5, wherein the haploid tissue is excised haploid tissue,the overexpressing step (c) is achieved in excised haploid tissuecultured in the presence of the inducer specific for the induciblepromoter, for a time sufficient to induce formation of the haploidsomatic embryo, followed by withdrawal of the inducer, and furtherwherein the growing step (d) is carried out in the absence of theinducer.